Department of Anatomy and Convergence Medical Science, Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju 52727, Korea.
Department of Obstetrics and Gynecology, College of Medicine, Gyeongsang National University, Jinju 52727, Korea.
Mol Cells. 2021 Mar 31;44(3):146-159. doi: 10.14348/molcells.2021.0001.
DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.
DNA 甲基化及其导致的肿瘤抑制基因下调，是癌症发展过程中对表观遗传刺激的反应。同样，人类致癌病毒，包括人乳头瘤病毒（HPV），上调并增强 DNA 甲基转移酶（DNMT）和组蛋白去乙酰化酶（HDAC）的活性，从而降低肿瘤抑制基因（TSGs）的表达。含泛素样结构域 PHD 和环指结构域 1（UHRF1）是 DNA 甲基化的表观遗传调节剂，在 HPV 诱导的宫颈癌中过度表达。在这里，我们通过慢病毒编码短发夹（sh）RNA 敲低 HeLa 细胞中的 UHRF1 表达，并进行 cDNA 微阵列分析，研究了 UHRF1 在宫颈癌中的作用。我们检测到 UHRF1 敲低细胞中硫氧还蛋白相互作用蛋白（TXNIP）的表达显著升高，这是一种已知的 TSG，全基因组甲基化分析表明该基因在宫颈癌组织和细胞系中呈高甲基化状态。Western blot 和免疫组化进一步检测到宫颈癌中 UHRF1 的上调和 TXNIP 的下调，并通过 Oncomine 数据库分析得到证实。通过染色质免疫沉淀，我们鉴定了 UHRF1 结合位点的倒位 CCAAT 结构域，并证明 UHRF1 敲低通过该区域的去甲基化降低 UHRF1 启动子结合并增强 TXNIP 表达。焦磷酸测序和甲基特异性聚合酶链反应进一步证实了宫颈癌组织中 启动子 CpG 甲基化。至关重要的是，UHRF1 的 siRNA 下调或 UHRF1 拮抗剂（百里醌）诱导细胞周期停滞和细胞凋亡，而 HPV 病毒蛋白 E6/E7 过表达会增加稳定和促进 UHRF1 功能的泛素特异性蛋白酶 7（USP7）。这些结果表明，HPV 可能通过 UHRF1 介导的 启动子甲基化诱导致癌作用，因此提示 CpG 甲基化与宫颈癌之间可能存在联系。
